The outer membrane of microorganisms includes various protein structures, such as flagella, pili, fimbriae, or protein capsules, which in particular endow the said microorganisms with properties of motion or attachment.
These structures include highly antigenic molecules that have made it possible to prepare, starting with the protein structure isolated from these microorganisms or from the microorganisms themselves, vaccines that correspond to the various types of antigens isolated in the microorganisms. Thus, the French patent application published under No. 2 466 251 proposes an anti-colibacillus animal vaccine that contains at least one antigen obtained from various strains of Escherichia coli.
Furthermore, these molecules constitute a comparable number of carrier proteins into which foreign epitopes can be introduced that endow the said proteins with a new antigenic nature, whose immunogenicity allows the said proteins to be used as vaccines. Thus, the European patent application published under No. 264 150 describes microorganisms whose outer membrane carries pili whose composition has been modified by a change in the protein sequence of the sub-unit.
Membrane structures such as pili, fimbriae, or protein capsules appear to be more advantageous in the field of vaccination than outer-membrane proteins. In fact, for pili, all of the structural protein is located outside the cell; certain polypeptide regions are present only at the cell surface; and accessibility to these proteins risks being impeded by the lipopolysaccharide and the capsular envelopes. Furthermore, the sub-units of the fimbriae or of the protein capsules are present at the bacterial surface in much greater numbers than the outer-membrane proteins.
Furthermore, the purification of a membrane protein is more difficult than the purification of proteins that are entirely outside the microorganisms, such as pili or fimbriae.
The nature of the microorganisms involved in the invention depends solely on their ability to produce the CS31A protein capsule. Specifically, it involves bacteria in the family of Enterobacteriaceae that belong to the Escherichia coli and Klebsiella pneumoniae species.
Many studies have been made of the pili of E. coli, and, more specifically, the K88 and K99 fimbrial sub-units have been amply described.
More recent studies have revealed, in a wild strain of E. coli designated as "31A+" in the French patent application published under No. 2 466 251, a protein structure designated as "CS31A". The operon that governs the biogenesis of this structure has been cloned in a host organism, and the gene that codes for the CS31A sub-unit, referred to as "Clpg", has been located, characterized, and sequenced. (See C. Martin, C. Boeuf, and F. Bousquet, in Microbial Pathogenesis, Vol. 10 (1991), pp. 429-442; J. P. Girardeau, Y. Bertin, C. Martin, M. DerVartanian, and C. Boeuf, in Journal of Bacteriology, Vol. 173, No. 23 (December 1991), pp. 7673-7983; and M. J. Korth, R. A. Schneider, and S. L. Moseley, in Infection and Immunity, Vol. 59, No. 7 (July 1991), pp. 2333-2340).
The CS31A structure is a protein capsule that is less organized and more flexible than the flagella and fimbriae, which are more organized and rigid. Consequently, it appears that the size of the peptides that can being expressed in the fimbriae and flagella could only with difficulty exceed approximately fifteen amino acids, whereas use of the CS31A protein capsules makes it possible to introduce larger foreign sequences, containing up to about a hundred amino acids.
Nevertheless, the introduction of heterologous peptide sequences is possible in the protein only at certain permissive sites, whose size and position must be determined.